1. Field of the Invention
The present invention relates to binary probes for fluorescent analysis of nucleic acids using molecular beacons or dyes.
2. Description of the Related Art
Sequence-specific detection of nucleic acids is crucial to disease diagnosis, genome study, and mRNA monitoring in living cells. Among the numerous methods for nucleic acid analysis are those that provide an immediate visible or fluorescent response after hybridization of the probe to complementary nucleic acid analytes. This offers easy and instant detection of the specific DNA and RNA analyte. However, the selectivity and efficacy of known methods is limited under physiological conditions, and this limitation hinders using the probes in living cells.
Numerous techniques for DNA and RNA analysis such as fluorescence in situ hybridization, micro-array technology, the molecular beacon approach and others rely on the ability of the probe to recognize DNA and RNA analytes in a sequence specific manner by forming duplexes. The formation of 16-20 nucleotide hybrids between probe and nucleic acid analyte is required in order to uniquely define a specific fragment in DNA the size of a genome. However, 16-20 nucleotide hybrid duplexes are too stable to be sensitive to a single mismatch at mild conditions. A number of different strategies have been developed in an attempt to solve this problem, however none effectively combine high sensitivity with mild conditions. Therefore, there is a need for probes that reliably detect single nucleotide mismatches (Single Nucleotide Polymorphisms, SNP) under milder or physiologic conditions.